Umadevi V. Wesley

Classification of Clear-Cell Sarcoma as a Subtype of Melanoma by Genomic Profiling

PURPOSE To develop a genome-based classification scheme for clear-cell sarcoma (CCS), also known as melanoma of soft parts (MSP), which would have implications for diagnosis and treatment. This tumor displays characteristic features of soft tissue sarcoma (STS), including deep soft tissue primary location and a characteristic translocation, t(12;22)(q13;q12), involving EWS and ATF1 genes. CCS/MSP also has typical melanoma features, including immunoreactivity for S100 and HMB45, pigmentation, MITF-M expression, and a propensity for regional lymph node metastases. MATERIALS AND METHODS RNA samples from 21 cell lines and 60 pathologically confirmed cases of STS, melanoma, and CCS/MSP were examined using the U95A GeneChip (Affymetrix, Santa Clara, CA). Hierarchical cluster analysis, principal component analysis, and support vector machine (SVM) analysis exploited genomic correlations within the data to classify CCS/MSP. RESULTS Unsupervised analyses demonstrated a clear distinction between STS and melanoma and, furthermore, showed that CCS/MSP cluster with the melanomas as a distinct group. A supervised SVM learning approach further validated this finding and provided a user-independent approach to diagnosis. Genes of interest that discriminate CCS/MSP included those encoding melanocyte differentiation antigens, MITF, SOX10, ERBB3, and FGFR1. CONCLUSION Gene expression profiles support the classification of CCS/MSP as a distinct genomic subtype of melanoma. Analysis of these gene profiles using the SVM may be an important diagnostic tool. Genomic analysis identified potential targets for the development of therapeutic strategies in the treatment of this disease.

Airway Epithelial Cell Migration and Wound Repair by ATP-mediated Activation of Dual Oxidase 1

The airway epithelium is continuously subjected to environmental pollutants, airborne pathogens, and allergens and relies on several intrinsic mechanisms to maintain barrier integrity and to promote epithelial repair processes following injury. Here, we report a critical role for dual oxidase 1 (Duox1), a newly identified NADPH oxidase homolog within the tracheobronchial epithelium, in airway epithelial cell migration and repair following injury. Activation of Duox1 during epithelial injury is mediated by cellular release of ATP, which signals through purinergic receptors expressed on the epithelial cell surface. Purinergic receptor stimulation by extracellular ATP is a critical determinant of epithelial cell migration and repair following injury and is associated with activation of extracellular signal-regulated kinases (ERK1/2) and matrix metalloproteinase-9 (MMP-9). Stimulation of these integral features of epithelial cell migration and repair processes was found to require the activation of Duox1. Our findings demonstrate a novel role for Duox1 in the tracheobronchial epithelium, in addition to its proposed role in antimicrobial host defense, by participating in epithelial repair processes to maintain epithelial integrity and barrier function in the face of environmental stress.

Role for dipeptidyl peptidase IV in tumor suppression of human non small cell lung carcinoma cells

Lung cancer is the leading cause of cancer death. Lung cancers produce a variety of mitogenic growth factors that stimulate tumor cell proliferation and migration. The cell surface protease, dipeptidyl peptidase IV (DPPIV), is involved in diverse biologic functions, including peptide‐mediated cellular growth and differentiation. DPPIV is expressed in various normal tissues, including lung tissue, and its expression is lost in many types of human cancers. DPPIV expression and its enzymatic activity are detected in normal bronchial and alveolar epithelium but different histologic subtypes of lung carcinomas lose DPPIV expression. To investigate the role of DPPIV in lung carcinoma, we examined the expression of DPPIV at both mRNA and protein levels in non small cell lung cancer (NSCLC) cell lines and normal human bronchial epithelial cells. DPPIV expression was detectable in normal lung epithelial cells, but was absent or markedly reduced in all NSCLC cell lines at both mRNA and protein levels. Restoration of DPPIV expression in NSCLC cells resulted in profound morphologic changes, inhibition of cell proliferation, anchorage‐independent growth, in vitro cell migration and tumorigenicity in nude mice. DPPIV reexpression also correlated with increased p21 expression, leading to induction of apoptosis and cell cycle arrest in G1 stage. These effects were accompanied by increased expression of cell surface proteins, fibroblast‐activating protein (Fapα) and CD44 that are associated with suppression of tumor growth and metastasis. Thus, DPPIV functions as a tumor suppressor, and its downregulation may contribute to the loss of growth control in NSCLC cells.

ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17

The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H2O2 participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y2 receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y2R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y2R activation as a proximal step in EGFR transactivation and downstream signaling.

Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1α and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1α stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1α activation. Activation of HIF-1α by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1α mutant construct inhibited epithelial wound repair, implicating HIF-1α in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1α. Finally, NO-mediated inhibition of cell migration was associated with HIF-1α-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1α and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.

Differential aminoacylase expression in neuroblastoma.

Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children. MYCN amplification and increased BDNF/TrkB signaling are features of high‐risk tumors; yet, only ˜25% of malignant tumors display these features. Thus, the identification of additional biomarkers and therapeutic targets is essential. As aminoacylase 1 (ACY1), an amino acid deacetylase, is a putative tumor suppressor in small cell lung and renal cell carcinomas, we investigated whether it or the other family members aspartoacylase (ASPA, aminoacylase 2) or aminoacylase 3 (ACY3) could serve a similar function in neuroblastoma. Aminoacylase expression was examined in TrkB‐positive, MYCN‐amplified (SMS‐KCNR and SK‐N‐BE) and TrkB‐negative, non‐MYCN‐amplified (SK‐N‐AS, SK‐N‐SH, SH‐SY5Y and SH‐EP) neuroblastoma cell lines. Each aminoacylase exhibited distinct spatial localization (i.e., cytosolic ACY1, membrane‐associated ASPA and nuclear ACY3). When SK‐N‐SH cells were treated with neural differentiation agents (e.g., retinoic acid and cAMP) in media containing 10% serum, ACY1 was the only aminoacylase whose expression was upregulated. ASPA was primarily expressed in SH‐EP cells of a glial sublineage. ACY3 was more highly expressed in the TrkB‐positive, MYCN‐amplified lines. All three aminoacylases were expressed in normal human adrenal gland, a common site of neuroblastoma origin, but only ACY1 and ACY3 displayed detectable expression in primary neuroblastoma tumor. Bioinformatics data mining of Kaplan–Meier survival revealed that high ACY3 expression is correlated with poor prognosis, whereas low expression of ACY1 or ASPA is correlated with poor prognosis. These data suggest that aminoacylase expression is dysregulated in neuroblastoma.

Evidence for the involvement of dominant-negative Notch molecules in the normal course of Drosophila development.

Notch signaling is used to specify cell types during animal development. A high level specifies one cell type, whereas a low level specifies the alternate type. The effector of Notch signaling is the Notch intracellular domain. Upon its release from the plasma membrane in response to Delta binding the Notch extracellular domain, the Notch intracellular domain combines with the transcription factor Suppressor of Hairless and promotes the expression of target genes. Using a panel of antibodies made against different extracellular and intracellular regions of Notch, we show that cell types and tissues with low levels of Notch signaling are enriched for Notch molecules detected only by the extracellular domain antibodies. This enrichment often follows enrichment for Notch molecules detected only by antibodies made against the Suppressor of Hairless binding region. Notch molecules lacking most of the intracellular domain or containing only the Suppressor of Hairless binding region are produced during development. Such molecules are known to suppress Notch signaling, possibly by taking away Delta or Suppressor of Hairless from the full‐length Notch. Thus, it is possible that dominant‐negative Notch molecules are produced in the normal course of tissue differentiation in Drosophila as part of an auto–down‐regulation mechanism. Developmental Dynamics 235:411–426, 2006.